Novel proteins, gene encoding the same and method of utilization thereof

ABSTRACT

The present invention provides, as a gene encoding an antigen recognized by G-CSF-inducing antibodies, a gene encoding: (a) a protein having the amino acid sequence listed as SEQ ID NO:2 of the Sequence Listing; (b) a protein having the amino acid sequence listed as SEQ ID NO:2 of the Sequence Listing with one or more amino acid deletions, substitutions, additions or insertions and also binding to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor; or (c) a protein having at least 50% homology with the amino acid sequence listed as SEQ ID NO:2 and also binding to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor.

This application is a Divisional of co-pending application Ser. No.09/937,905, filed on Oct. 1, 2001, and for which priority is claimedunder 35 U.S.C. § 120. application Ser. No. 09/937,905 is the nationalphase of PCT International Application No. PCT/JP00/02080 under 35U.S.C. § 371, which has an International filing date of Mar. 31, 2000,and which claims priority from Japanese Application No. 95092/1999 filedon Apr. 1, 1999. The entire contents of each of the above-identifiedapplications are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a protein which is reactive withantibodies that are active to induce granulocyte colony-stimulatingfactor, to a gene encoding it and to a method for their use.

BACKGROUND ART

Granulocyte colony-stimulating factor (G-CSF) has a molecular weight ofapproximately 18,000 to 22,000 and consists of 174 (in rare cases 178)amino acids in the case of humans and 178 amino acids in the case ofmice. It is a glycoprotein that induces differentiation andproliferation of neutrophils, one of the types of leukocytes.

G-CSF has a potential of survival-extension and functional promotion tothe mature neutrophils, and

also has ability to form erythroblasts in response to erythropoietin andblast cell colonies in response to interleukin-3. Cells that produceG-CSF are macrophages, stroma cells, monocytes, T lymphocytes,fibroblasts, vascular endothelial cells and so forth.

Administration of G-CSF drug exhibits a therapeutic effect onneutropenia induced by side effect of anticancer agents, or neutropeniafollowing bone marrow transplantation, and a therapeutic effect onanaplastic anemia. Because of its low stability in the blood, however,it requires frequent administration, and because its administration islimited to the intravenous route, this has resulted in a great deal ofpain and burden to the patient and physician. Furthermore,administration of G-CSF as a drug has been reported to cause ostalgia asa side-effect. The alternative option of direct administration ofmacrophages or stroma cells that produce G-CSF will produce the risk ofunknown side-effects since the cells contain numerous proteins and othersubstances, and therefore such treatment has not been practiced.

Because administration of G-CSF itself for differentiation andproliferation of neutrophils provoke ostalgia as a side-effect, and italso requires frequent administration and increases the pain and burdento the patient and physician, it has been strongly desired to develop analternative treatment method; however, no such method has yet beenestablished.

With the intent of causing production of G-CSF and differentiation andproliferation of neutrophils without administration of G-CSF itself, thepresent inventors have already succeeded in providing G-CSF inducingantibodies (Japanese Patent Application HEI No. 9-266591 (Sep. 30,1997), Japanese Unexamined Patent Publication HEI No. 11-106400 (Apr.20, 1999)).

However, the antigens recognized by the G-CSF inducing antibodies havenot yet been discovered.

One problem to be solved by the present invention, therefore, is toidentify an antigen recognized by G-CSF inducing antibodies. Anotherproblem to be solved by the invention is to clone and identify the geneencoding the antigen recognized by the G-CSF inducing antibodies.

DISCLOSURE OF THE INVENTION

As a result of diligent research aimed at solving the problems describedabove, the present inventors used monoclonal antibodies with G-CSFinducing ability as

probes for immunoscreening of a cDNA library derived from macrophagecells, and as a result succeeded in isolating 3 positive clones, andthen further determined the nucleotide sequences thereof, to thus arriveat the present invention. The present inventors also determined thenucleotide sequence of the human antigen gene.

In other words, the present invention provides a gene encoding: (a) aprotein having the amino acid sequence listed as SEQ ID NO:2 of theSequence Listing; (b) a protein having the amino acid sequence listed asSEQ ID NO:2 of the Sequence Listing with one or more amino aciddeletions, substitutions, additions or insertions and also binding to anantibody or its fragment that is active to induce granulocytecolony-stimulating factor; or (c) a protein having at least 50% homologywith the amino acid sequence listed as SEQ ID NO:2 and also binding toan antibody or its fragment that is active to induce granulocytecolony-stimulating factor.

The invention further provides a gene encoding: (a) a protein having theamino acid sequence listed as SEQ ID NO:4 of the Sequence Listing; (b) aprotein having the amino acid sequence listed as SEQ ID NO:4 of theSequence Listing with one or more amino acid deletions, substitutions,additions or insertions and also binding to an antibody or its fragmentthat is active to induce granulocyte colony-stimulating factor; or (c) aprotein having at least 50% homology with the amino acid sequence listedas SEQ ID NO:4 and also binding to an antibody or its fragment that isactive to induce granulocyte colony-stimulating factor.

The invention further provides a gene having: (a) the nucleotidesequence listed as SEQ ID NO:1 of the Sequence Listing; (b) a nucleotidesequence which is the nucleotide sequence listed as SEQ ID NO:1 of theSequence Listing with one or more nucleotide deletions, substitutions,additions or insertions and which encodes a protein that can bind to anantibody or its fragment that is active to induce granulocytecolony-stimulating factor; or (c) a nucleotide sequence which hybridizeswith DNA having the nucleotide sequence listed as SEQ ID NO:1 of theSequence Listing under stringent conditions and encodes a protein thatcan bind to an antibody or its fragments that are active to inducegranulocyte colony-stimulating factor.

The invention further provides a gene having: (a) the nucleotidesequence listed as SEQ ID NO:3 of the Sequence Listing; (b) a nucleotidesequence which is the nucleotide sequence listed as SEQ ID NO:3 of theSequence Listing with one or more nucleotide deletions, substitutions,additions or insertions and which encodes a protein that can bind to anantibody or its fragment that is active to induce granulocytecolony-stimulating factor; or (c) a nucleotide sequence which hybridizeswith DNA having the nucleotide sequence listed as SEQ ID NO:3 of theSequence Listing under stringent conditions and encodes a protein thatcan bind to an antibody or its fragment that is active to inducegranulocyte colony-stimulating factor.

The antibody that is active to induce granulocyte colony-stimulatingfactor mentioned above is, for example, the monoclonal antibody producedby a hybridoma of the cell line deposited as FERM BP-6103.

According to the invention, the gene is a gene derived from a mouse orhuman.

The invention further provides a DNA fragment containing: (1) thenucleotide sequence from position 519 to position 736, the nucleotidesequence from position 666 to position 689, the nucleotide sequence fromposition 381 to position 403 or the nucleotide sequence from position709 to position 727 of the nucleotide sequence listed as SEQ ID NO:1 ofthe Sequence Listing; (2) a nucleotide sequence which is any of thenucleotide sequences of (1) above with one or more nucleotide deletions,substitutions, additions or insertions; or (3) a nucleotide sequencewhich has at least 80% homology with any of the nucleotide sequences of(1) above.

The invention further provides a gene containing: (1) the nucleotidesequence from position 519 to position 736, the nucleotide sequence fromposition 666 to position 689, the nucleotide sequence from position 381to position 403 or the nucleotide sequence from position 709 to position727 of the nucleotide sequence listed as SEQ ID NO:1 of the SequenceListing; (2) a nucleotide sequence which is any of the nucleotidesequences of (1) above with one or more nucleotide deletions,substitutions, additions or insertions; or (3) a nucleotide sequencewhich has at least 80% homology with any of the nucleotide sequences of(1) above; and encoding a protein that can bind to an antibody or itsfragments that are active to induce granulocyte colony-stimulatingfactor.

The invention further provides any of the following proteins: (a) aprotein having the amino acid sequence listed as SEQ ID NO:2 of theSequence Listing; (b) a protein having the amino acid sequence listed asSEQ ID NO:2 of the Sequence Listing with one or more amino aciddeletions, substitutions, additions or insertions and also binding to anantibody or its fragment that is active to induce granulocytecolony-stimulating factor; (c) a protein having at least 50% homologywith the amino acid sequence listed as SEQ ID NO:2 and also binding toan antibody or its fragment that is active to induce granulocytecolony-stimulating factor; or (d) a protein that is encoded by DNA whichhybridizes with DNA having the nucleotide sequence listed as SEQ ID NO:1of the Sequence Listing under stringent conditions and that binds to anantibody or its fragments that are active to induce granulocytecolony-stimulating factor.

The invention further provides any of the following proteins: (a) aprotein having the amino acid sequence listed as SEQ ID NO:4 of theSequence Listing; (b) a protein having the amino acid sequence listed asSEQ ID NO:4 of the Sequence Listing with one or more amino aciddeletions, substitutions, additions or insertions and also binding to anantibody or its fragment that is active to induce granulocytecolony-stimulating factor; (c) a protein having at least 50% homologywith the amino acid sequence listed as SEQ ID NO:4 and also binding toan antibody or its fragment that is active to induce granulocytecolony-stimulating factor; or (d) a protein that is encoded by DNA whichhybridizes with DNA having the nucleotide sequence listed as SEQ ID NO:3of the Sequence Listing under stringent conditions and that binds to anantibody or its fragment that is active to induce granulocytecolony-stimulating factor.

The antibody that is active to induce granulocyte colony-stimulatingfactor mentioned above is, for example, the monoclonal antibody producedby a hybridoma of the cell line deposited as FERM BP-6103.

According to the invention, the protein is preferably a protein derivedfrom mammals and most preferably from a mouse or human.

The invention further provides a protein comprising any of thefollowings: (1) the amino acid sequence from residues 1 to 91, the aminoacid sequence from residues 50 to 146, the amino acid sequence fromresidues 1 to 78, the amino acid sequence from residues 200 to 241, theamino acid sequence from residues 172 to 241, the amino acid sequencefrom residues 103 to 150 or the amino acid sequence from residues 169 to241 of the amino acid sequence listed as SEQ ID NO:2 of the SequenceListing; (2) an amino acid sequence which is any of the amino acidsequences of (1) above with one or more amino acid deletions,substitutions, additions or insertions; or an amino acid sequence havingat least 70% homology with any of the amino acid sequences of (1) above.

The invention further provides a protein comprising any of thefollowings: (1) the amino acid sequence from residues 1 to 91, the aminoacid sequence from residues 50 to 146, the amino acid sequence fromresidues 1 to 78, the amino acid sequence from residues 200 to 241, theamino acid sequence from residues 172 to 241, the amino acid sequencefrom residues 103 to 150 or the amino acid sequence from residues 169 to241 of the amino acid sequence listed as SEQ ID NO:2 of the SequenceListing; (2) an amino acid sequence which is any of the amino acidsequences of (1) above with one or more amino acid deletions,substitutions, additions or insertions; or (3) an amino acid sequencehaving at least 70% homology with any of the amino acid sequences of (1)above, and also binding to an antibody or its fragments that are activeto induce granulocyte colony-stimulating factor.

The invention further provides an antibody or fragment thereof, for theaforementioned protein of the invention. The antibody is preferably amonoclonal antibody, and most preferably a human-type monoclonalantibody or human monoclonal antibody.

The invention further provides a recombinant vector containing a gene ora DNA fragment according to the invention.

The invention further provides a transformant comprising a recombinantvector containing a gene or a DNA fragment according to the invention.

The invention further provides a receptor for a substance that caninduce production of granulocyte colony-stimulating factor, comprising aprotein according to the invention.

The invention further provides a method employing a protein of theinvention for screening of a useful substance (for example, an agonistor antagonist for the protein), the substance obtained by the screeningmethod and a useful substance that can bind to a receptor (for example,an agonist or antagonist for the receptor).

The invention further provides a pharmaceutical compounds comprising agene, a DNA fragment, a protein (or a protein fragment), an antibody (oran antibody fragment), a receptor or a substance according to theinvention (particularly a pharmaceutical compounds for diagnosis,prevention or treatment of infectious diseases or neutropenia), and atreatment method employing it.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments and working methods of the invention will now be explainedin detail.

Prior to the present invention, the present inventors succeeded inobtaining antibodies by direct immunization of macrophages and isolatingG-CSF inducing antibodies among the obtained antibodies (Japanese PatentApplication HEI No. 9-266591, all of the content of which isincorporated in the present specification by reference). The gene of thepresent invention was isolated by using these antibodies as a probe forscreening of a cDNA library derived from mouse macrophages, and theprotein encoded by the gene of the invention is characterized by theability to bind to the antibodies or the fragments thereof which havegranulocyte colony-stimulating factor-inducing activity.

(Antibodies or Fragments Thereof which have GranulocyteColony-Stimulating Factor-Inducing Activity)

First, an explanation will be provided regarding the method of obtainingthe “antibodies or fragments which have granulocyte colony-stimulatingfactor-inducing activity” according to the invention. (Also referred toas “antibodies used for the invention” hereinafter throughout thepresent specification.)

The present inventors administered a mouse macrophage cell line as animmunogen to MRL/lpr mice (autoimmune mice), and isolated monoclonalantibodies. Then, the obtained monoclonal antibodies were applied to themouse macrophage cell line and examined the effect of the antibodies tothe macrophage cells. As a result, it was discovered that one of theobtained antibodies had the character of causing production of G-CSF bythe mouse macrophage cell line in a concentration-dependent manner. (Thehybridoma cell line that produces the antibody has been deposited asFERM BP-6103.)

Throughout the present specification, the term “monoclonal antibodies”will refer to monoclonal antibodies with reactivity to the macrophagecell line, and specifically refer to monoclonal antibodies with afunction of causing production of G-CSF.

The antibodies used for the invention had the character to bindsubstantially to the macrophage cell line. The antibodies used for theinvention include any polyclonal antibody or monoclonal antibody havingthis characters. The “monoclonal antibodies” include monoclonalantibodies belonging to all the immunoglobulin classes IgG, IgM, IgA,IgD and IgE, and are preferably monoclonal antibodies of theimmunoglobulin classes IgG and IgM.

The macrophage cell line may be prepared from naturally occurringleukemia cells or it may be prepared by transformation with leukemiavirus.

The antibodies used for the invention may be obtained according to acommon procedure (for example, the method described in “Zoku SeikagakuJikken Koza 5, Men'eki Seikagaku Kenkyuuhou” [Methods of BiochemicalExperiments V—Immunobiochemistry Research Methods], ed. by The JapaneseBiochemical Society: published by Tokyo Kagaku Dojin).

The monoclonal antibodies used for the invention can be produced from ahybridoma (fused cell line) created by cell fusion. The hybridoma thatproduces monoclonal antibodies is prepared by the following: theantibody-producing cells are fused with myeloma cells, and then theantibody-producing hybridoma is cloned by specific binding to themacrophage cell line. This procedure may be carried out according toconventionally known protocols except for the use of all or part of themacrophage cell line as the immunogen.

The immunogen used may be the macrophage cell line itself, or else theremay be prepared a (poly)peptide solutions derived from all or a portionof the membrane fraction or soluble extract of the macrophage cell line,or a mixture thereof with Freund's complete adjuvant.

The animals used as the object of immunization may be a mammal such as amouse, rat, guinea pig, hamster or rabbit, and is preferably a mouse orrat, and more preferably a mouse. The immunization is carried out by oneor several injections into the mammals through a subcutaneous,intramuscular, intravenous, foot pad or intraabdominal route.

Following initial immunization, 1-4 booster immunizations were carriedout every 1-2 weeks interval, with a final immunization after another1-4 weeks. The antibody-producing cells are collected from theimmunosensitized animals after about 3-5 days from the finalimmunization.

The monoclonal antibodies used for the invention include the monoclonalantibodies produced by a hybridoma of “FERM BP-6103” (3-4H7 antibodies),their fragments and antibodies having essentially the same characters.“3-4H7 antibody” has the ability to induce G-CSF production by thecells.

The hybridoma that produces the monoclonal antibodies used for theinvention may be prepared by a commonly known method. As an example of acommonly known method for preparation of a monoclonal antibody-secretinghybridoma, there may be mentioned in the methods of Koehler and Milstein(Nature, Vol. 256, pp. 495-497, 1975) or its modified methods. Here, themonoclonal antibodies are prepared by culturing fused cells (ahybridoma) that are obtained by fusing antibody-producing cells from aspleen, lymph node, bone marrow or tonsil, preferably a spleen, takenfrom an animal immunosensitized as described above, with myeloma cellsfrom a mammalian animal such as a mouse, rat, guinea pig, hamster,rabbit or human, preferably of the same species, and more preferablyfrom a mouse, rat, or human. The culturing may be carried out in vitro,or in vivo in the ascites fluid of a mammalian animal such as a mouse,rat, guinea pig, hamster or rabbit, preferably a mouse or rat, and morepreferably a mouse, and the antibodies may be obtained from each culturesupernatants or from the ascites fluid of the mammalian animal.

As examples of myeloma cell lines to be used for the cell fusion, theremay be the mouse-derived myelomas “P3/X63-AG8”, “P3/NSI/1-Ag4-1”,“P3/X63-Ag8.U1”, “SP2/0-Ag14”, “PAI”, “FO” or “BW5147”, the rat-derivedmyeloma “210RCY3-Ag1.2.3” and the human-derived myelomas “U-266AR1”,“GM1500-6TG-A1-2”, “UC729-6”, “CEM-AGR”, “D1R11” and “CEM-T15”.

The screening of fused cell clones that produce the monoclonalantibodies and used for the invention may be accomplished by measuringthe antigen reactivity of the culture supernatants from the wellsexhibiting cell growth in a microtiter plate using anenzyme-immunological method such as flow cytometry, RIA or ELISA.

As examples of basic media, there may be low calcium media such asHam'F12 medium, MCDB153 medium or low calcium MEM medium, and highcalcium media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium. Serum, hormone cytokines and/orvarious organic or inorganic substances may be added to the basic mediumdepending on the purpose. Isolation and purification of the monoclonalantibodies from the culture supernatant or ascites fluid can beaccomplished by various method such as saturated ammonium sulfatemethod, euglobulin precipitation, caproic acid method, caprylic acidmethod, ion-exchange chromatography (DEAE, DE52, etc.), affinity columnchromatography with an anti-immunoglobulin column or Protein A orProtein G column, or hydrophobic chromatography.

The monoclonal antibodies used for the invention may also be obtained byany other method, without being restricted to the production methoddescribed above. Ordinary “monoclonal antibodies” have sugar chains withdifferent structures depending on the types of a mammalian animal whichhas been immunosensitized, and the “monoclonal antibodies” used for theinvention are not limited by structural differences in these sugarchains and include any monoclonal antibody derived from mammaliananimals. The “monoclonal antibodies” used for the invention also includemonoclonal antibodies produced by phage display, as well as human-typemonoclonal antibodies obtained using transgenic mice created by geneticengineering that produce human-type antibodies by incorporation of humanimmunoglobulin genes, for example, chimeric monoclonal antibodiesobtained by using gene recombination techniques to recombine theconstant region (Fc region) of a mammalian animal-derived monoclonalantibody with the Fc region of a human monoclonal antibody, andhumanized monoclonal antibodies obtained by recombining thecomplementarity-determining region (CDR) that can directly bind in acomplementary manner with the antigen, with the corresponding region ofa human monoclonal antibody.

According to the invention, an “antibody fragment” may also be used,where “antibody fragment” means an antibody fragment including at leastone variable region, synonymous with the “antibody portion” mentioned inJapanese Patent Application HEI No. 9-266591. Specifically, it refers tothe Fv, F(ab′)2, Fab′ or Fab fragments. Here, “F(ab′)2” and “Fab′” referto antibody fragments produced by treating an immunoglobulin (monoclonalantibody) with a protease such as pepsin or papain, and they areobtained by digestion before and after the sulfide bonds present betweenthe two H chains at the hinge region. For example, treatment of IgG withpapain cleaves it upstream from the disulfide bonds present between thetwo H chains at the hinge region, producing two homologous antibodyfragments each comprising an L chain composed of a VL (L chain variableregion) and CL (L chain constant region) and an H chain fragmentcomposed of a VH (H chain variable region) and CHγ1 (γ1 region of the Hchain constant region) which are bonded by sulfide bonds at theC-terminal region. These two homologous antibody fragments are eachdesignated as Fab′. Treatment of IgG with pepsin cleaves it downstreamfrom the disulfide bonds present between the two H chains at the hingeregion, producing an antibody fragment which is slightly larger thanjust the aforementioned two Fab′ fragments connected at the hingeregion. This antibody fragment is designated as F(ab′)2.

The protein encoded by the gene of the invention is characterized bybinding to an antibody or its fragment that is active to inducegranulocyte colony-stimulating factor, as explained in detail above. Theterm “binding” as used throughout the present specification meansordinary binding between a protein and antibody, and it may be measuredusing common immunological analysis techniques (for example,immunoprecipitation, ELISA, immunoblotting, etc.)

(Gene of the Invention)

The present invention provides a gene which encodes the protein havingthe amino acid sequence listed as SEQ ID NO:1 of the Sequence Listing ora protein which is homologous thereto. The invention also provides agene having the nucleotide sequence listed as SEQ ID NO:1 of theSequence Listing or a nucleotide sequence which is homologous thereto.

There are no particular restrictions on the type of the gene of theinvention, and it may be naturally occurring DNA, recombinant DNA orchemically synthesized DNA, or even a genomic DNA clone or a cDNA clone.

The gene of the invention will typically have the nucleotide sequencelisted as SEQ ID NO:1 of the Sequence Listing, but this is only thesequence of a clone (MMR19) obtained in the following examples torepresent an embodiment of the invention. Those skilled in the art arewell aware that natural genes have a small number of variationsdepending on the breeding conditions of the biological species thatproduce it, and on the ecosystem, or on the presence of highly similarisozymes. Consequently, the “gene” of the invention is not limited tothe gene having the nucleotide sequence listed as SEQ ID NO:1 of theSequence Listing but also includes all genes encoding proteins havingthe characters described in the present specification.

Consequently, the “gene” of the invention is not limited to the genehaving the nucleotide sequence listed as SEQ ID NO:1 of the SequenceListing but also includes all genes encoding proteins having thecharacters described in the present specification.

In particular, disclosure by the present specification of the amino acidsequence for the protein of the invention and the DNA sequence encodingthe protein easily allows to isolate a gene encoding a protein withsimilar physiological activity from another biological species byutilizing basic genetic engineering techniques such as hybridization orPCR. Genes obtained in this manner are also within the scope of thepresent invention.

There are no particular restrictions for the hybridization conditionsused for screening of a homologous gene, and they may be appropriatelyselected by a person skilled in the art depending on the degree ofhomology between the target homologous gene and probe, althoughstringent conditions are generally preferred, and for example, thehybridization conditions may be 6×SSC [0.9 M NaCl, 0.09 M sodium citrate(pH 7.0)], 5×Denhardt's solution [1 g ficoll, 1 g polyvinyl pyrrolidone,1 g BSA in 1000 mL], 0.5% SDS, 25° C.-68° C. (for example, 37° C., 42°C. or 68° C.), or 0-50% formamide, 6×SSC, 0.5% SDS, 25-68° C. (forexample, 37° C., 42° C. or 68° C.). It is well known to those skilled inthe art that appropriate setting of the hybridization conditionsincluding the formamide concentration, Denhardt's solutionconcentration, salt concentration, temperature, etc. will allow to cloneDNA containing a nucleotide sequence with a given degree of homology orgreater, and all homologous genes cloned in this manner are within thescope of the present invention.

A homologous gene cloned by hybridization in this manner has at least70%, preferably at least 80%, more preferably at least 90%, even morepreferably at least 95% and most preferably at least 98% homology withrespect to the nucleotide sequence listed as SEQ ID NO:1 of the SequenceListing.

(Protein of the Invention)

The present invention provides the protein having the amino acidsequence listed as SEQ ID NO:1 of the Sequence Listing or a proteinhomologous thereto.

The protein having the amino acid sequence listed as SEQ ID NO:1 of theSequence Listing according to the invention may be obtained byincorporating the gene encoding therefor into an appropriate expressionvector, transforming this vectors to an appropriate host and expressingthe recombinant protein. However, the source and preparation method arenot restricted so long as the protein of the invention has thecharacters described in the present specification, and it may be anaturally produced protein, a protein expressed from recombinant DNA bya genetic engineering method or a protein chemically synthesized.

The protein of the invention will typically have the sequence of 241amino acids listed as SEQ ID NO:1 of the Sequence Listing. However,those skilled in the art are well aware that natural proteins includevariations of one or more amino acids due to gene variation depending onthe breeding conditions of biological species that produce it, on theecosystem, the presence of highly similar isozymes. The term “amino acidvariation” as used here means one or more amino acid substitutions,deletions, insertions and/or additions. The “protein” of the inventionhas the amino acid sequence listed as SEQ ID NO:1 based on deductionfrom the nucleotide sequence of the cloned gene, but it is not limitedonly to proteins with that sequence and is intended to include allhomologous proteins that have the characteristics described in thepresent specification. The homology is at least 50%, preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, yet more preferably at least 95% and mostpreferably at least 98%.

Generally speaking, introduction of a substitution between amino acidswith the same nature (for example, a substitution between twohydrophobic amino acids, a substitution between two hydrophilic aminoacids, a substitution between two acidic amino acids or a substitutionbetween two basic amino acids) will usually tend to give a variedprotein with the same characters as the original protein. Those skilledin the art are familiar with methods of preparing recombinant proteinswith desired characters using gene recombinant techniques, and suchvaried proteins are also within the scope of the invention.

The following examples in the present specification describe cloning ofmouse macrophage-derived cDNA as an embodiment of the invention. The useof the amino acid sequence of the protein disclosed in the presentspecification or the sequence of the (mouse-derived) gene codingtherefor, or a portion thereof, for isolation of a gene encoding aprotein from another source but having similar physiological activityusing gene engineering techniques such as hybridization or PCR, iswithin the scope of commonly accepted knowledge to a person skilled inthe art, and proteins encoded by such isolated genes are also within thescope of the present invention.

(Human-Type Gene and Protein)

For example, the following method may be mentioned as an example ofobtaining a human-derived homologue of the gene and protein of theinvention.

The total RNA is extracted from a human macrophage cell line (THP-1,U937, HL-60) by guanidium thiocyanate-phenol-chloroform single-stepextraction (Laboratory Manuals of Genetic Engineering, 3rd Edition, pp.83-84, 1996), and purified using an oligo(dT) cellulose column, toobtain poly(A)+ RNA. Reverse transcriptase (MMLV-RTase) and DNApolymerase are used to synthesize double-stranded cDNA. Thedouble-stranded cDNA is used to construct a cDNA library using a IZAPIIphage vector by the method of Gubler-Hoffmann (Gubler, U. and Hoffmann,B. J.: Gene, 25:263-269, 1983). A probe is then prepared by amplifying aDNA sequence using a primer DNA that can amplify a sequence in theregion of the nucleotide sequence (SEQ ID NO:1) of the mouse cDNA (MMR19clone) disclosed in the present specification, having high homology withthe human sequence (for example, the region from position 172 toposition 241 of SEQ ID NO:1 which has been found to have 91% homologywith the human sequence) and the template DNA from the human macrophagecell cDNA library. Or region (for example, the region from position 172to position 241 of SEQ ID NO:1) is used directly as the probe forscreening the cDNA encoding the entire length of the target protein fromthe human macrophage cell cDNA library. The cDNA nucleotide sequence isanalyzed by the Primer Walking method. The cDNA confirmed to encode theentire length of the target protein is introduced into a baculovirus toexpress a protein, which can be purified with an affinity column toobtain the human-type homologous protein.

As explained above, the present invention relates to the gene or proteinhaving the nucleotide sequence listed as SEQ ID NO:1 or the amino acidsequence listed as SEQ ID NO:2, and to genes and proteins which arehomologous thereto. As a result of a search to determine whether or notsequences homologous to the nucleotide sequence listed as SEQ ID NO:1and the amino acid sequence listed as SEQ ID NO:2 provided by theinvention are present in other organisms, it was confirmed that humanESTs (expressed sequence tags) include sequences having high homologywith the gene of the invention (see Example 3 below). It will thereforebe readily appreciated that the human-derived homologous gene can alsobe isolated by screening of a human-derived gene library (cDNA library,etc.) using the human-derived ESTs having high homology with thenucleotide sequence of the invention as the probe.

As described above, a database search revealed that portions (i.e. DNAfragments) of the nucleotide sequence listed as SEQ ID NO:1 according tothe invention are conserved, having high homology with the humansequence. Such DNA fragments are useful as probes for screening of thehuman-derived homologous gene, and therefore constitute one aspect ofthe present invention. The DNA fragments include DNA fragmentscontaining the nucleotide sequence from position 519 to position 736,the nucleotide sequence from position 666 to position 689, thenucleotide sequence from position 381 to position 403 or the nucleotidesequence from position 709 to position 727 of the nucleotide sequencelisted as SEQ ID NO:1 of the Sequence Listing, while DNA fragmentscontaining a nucleotide sequence which is any of these nucleotidesequences with one or more nucleotide deletions, substitutions,additions or insertions or a nucleotide sequence which has at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and most preferably at least 98% homology withany of these nucleotide sequences, are also within the scope of theinvention.

A database search also revealed that a portion of the amino acidsequence listed as SEQ ID NO:2 according to the invention is conservedwith high homology in the human sequence. Protein fragments comprisingportions of the protein of the invention are also useful as reagents foranalysis and isolation of antibodies with G-CSF inducing activity, as isthe protein of the invention, and also have potential utility as a druglike the protein of the invention, and thus constitute an aspect of theinvention.

The mentioned proteins include the amino acid sequences from residues 1to 91, 50 to 146, 1 to 78, 200 to 241, 172 to 241, 103 to 150, and 169to 241 of the amino acid sequence listed as SEQ ID NO:2 of the SequenceListing, while proteins containing an amino acid sequence which is anyof these amino acid sequences with one or more amino acid deletions,substitutions, additions or insertions or an amino acid sequence havingat least 70%, preferably at least 80%, more preferably at least 85%,even more preferably at least 90%, even more preferably at least 95% andmost preferably at least 98% homology with any of these amino acidsequences, are also within the scope of the invention.

The present inventors have determined the nucleotide sequence of thehuman-type antigen gene by a method similar to the one described above(see Example 4 below). Consequently, the present invention provides agene having the nucleotide sequence listed as SEQ ID NO:3 of theSequence Listing or having a nucleotide sequence which is homologousthereto. The invention also provides a protein having the amino acidsequence listed as SEQ ID NO:4 of the Sequence Listing or a proteinwhich is homologous thereto. Here, “homologous” means that the scope ofthe invention is not limited to the gene having the nucleotide sequencelisted as SEQ ID NO:3 or the protein having the amino acid sequencelisted as SEQ ID NO:4, as was explained in the part of “Gene of theinvention” and “Protein of the invention” described above.

(Antibodies of the Invention)

The invention also provides antibodies against the above-mentionedprotein of the invention (also referred to hereinafter in the presentspecification as “monoclonal antibodies of the invention”). An exampleof the antibodies of the invention and a method for obtaining them isexplained in detail.

The antibodies of the invention may be either polyclonal antibodies ormonoclonal antibodies, and in the case of monoclonal antibodies they maybe chimeric antibodies. Mouse/human chimeric antibodies are particularlypreferred. The “monoclonal antibodies” include monoclonal antibodiesbelonging to all the immunoglobulin classes such as IgG, IgM, IgA, IgDand IgE, and are preferably monoclonal antibodies of the immunoglobulinclasses IgG and IgM.

The protein of the invention used as the antigen may be obtained byincorporating a gene encoding it into an appropriate expression vector,transforming the incorporated vector to an appropriate host andexpressing the recombinant protein. The immunogen used may be, forexample, an actual macrophage cell line or the membrane fraction of amacrophage cell line.

The antibodies of the invention, such as polyclonal antibodies(antiserum) or monoclonal antibodies may be obtained according to acommon procedure (for example, the method described in “Zoku SeikagakuJikken Koza 5, Men'eki Seikagaku Kenkyuuhou” [Method of BiochemicalExperiments V—Immunobiochemistry Research Methods], ed. by The JapaneseBiochemical Society: published by Tokyo Kagaku Dojin).

Specifically, a mammalian animal, preferably a mouse, rat, hamster,guinea pig, rabbit, dog, cat, pig, goat, horse or cow, and morepreferably a mouse, rat, hamster, guinea pig or rabbit, is immunizedwith the antigen in combination with Freund's adjuvant if necessary.Polyclonal antibodies may be collected from serum obtained from theimmunosensitized animal. Monoclonal antibodies may be produced from ahybridoma made of fusions between antibody-producing cells obtained fromthe immunosensitized animal and a myeloma cell line (myeloma cells)which has no ability to produce antibodies. The hybridoma was cloned thehybridoma and selected clones that produce monoclonal antibodiesexhibiting specific affinity toward the antigen used to immunize themammalian animal.

Specifically, the monoclonal antibodies may be produced in the followingmanner. The protein of the invention or cells expressing the protein ofthe invention are used as the immunogen together with Freund's adjuvantif necessary. For immunosensitization, a mouse, rat, hamster, guinea pigor rabbit, and preferably a mouse, rat or hamster (such animals includetransgenic animals created to produce antibodies of other animals, suchas human antibody-producing transgenic mice) is used by one or severalinjections through a subcutaneous, intramuscular, intravenous, foot pador intraabdominal route, or by transplantation. Usually 1 to 4 boosterimmunizations are given every 1 to 14 days from the initialimmunization, and 1 to 5 days after the final immunization, and theantibody-producing cells are taken from the immunosensitized mammaliananimal.

The monoclonal antibodies of the invention may be produced from ahybridoma (fused cells) produced by cell fusion.

The hybridoma producing the monoclonal antibodies can be prepared by acommonly known method. As a commonly known method, it may be mentionedin the method of Koehler and Milstein (Nature, Vol. 256, pp. 495-497,1975) or methods with modifications of that method. Specifically, themonoclonal antibodies are prepared by culturing fused cells (ahybridoma), which are obtained by fusing antibody-producing cells from aspleen, lymph nodes, bone marrow or tonsils, preferably a spleen, takenfrom an animal immunosensitized in the manner described above, withmyeloma cells from a mammalian animal such as a mouse, rat, guinea pig,hamster, rabbit or human, and preferably from a mouse, rat, or human.

As examples of myeloma cell lines used for the cell fusion, there may bethe mouse-derived myelomas as mentioned above, such as “P3/X63-AG8”,“P3/NSI/1-Ag4-1”, “P3/X63-Ag8.U1”, “SP2/0-Ag14”, “X63,653”, “PAI”, “FO”or “BW5147”, the rat-derived myeloma “210RCY3-Ag1.2.3” and thehuman-derived myelomas “U-266AR1”, “GM1500-6TG-A1-2”, “UC729-6”,“CEM-AGR”, “D1R11” and “CEM-T15”.

Screening of fused cell clones that produce the monoclonal antibodiesused for the invention may be accomplished by culturing the fused cellsin a microtiter plate, for example, and by using flow cytometry, RIA,ELISA alternative the like to measure the antigen reactivity of theculture supernatants from the wells exhibiting growth.

Production of monoclonal antibodies from the hybridoma may be carriedout by culturing the hybridoma in vitro, or in vivo in the ascites fluidof a mouse, rat, guinea pig, hamster or rabbit, preferably a mouse orrat, and more preferably a mouse, and isolating the antibodies from theresulting culture supernatant or from the ascites fluid of the mammaliananimal. In the case of in vitro culture, the hybridoma may be grown,maintained and stored following a condition varied depending on theproperties of the cell line being cultured, the purpose of research andthe culturing method. And the culturing used for production ofmonoclonal antibodies in culture supernatants may be carried out using aknown nutrient medium or any nutrient medium derived and prepared from aknown basic medium.

As examples of basic media, there may be the low calcium media such asHam'F12 medium, MCDB153 medium or low calcium MEM medium, and highcalcium media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640medium, ASF104 medium or RD medium. Serum, hormone cytokines and/orvarious organic or inorganic substances may also be added to the basicmedium, depending on the purpose. Isolation and purification of themonoclonal antibodies can be accomplished by apply a saturated ammoniumsulfate method, euglobulin precipitation, caproic acid method, caprylicacid method, ion-exchange chromatography (DEAE, DE52, etc.), affinitycolumn chromatography with an anti-immunoglobulin column or Protein A orProtein G column, or else subjecting them to hydrophobic chromatographyto subjecting the culture supernatant or ascites fluid.

A “chimeric antibody” according to the invention is a monoclonalantibody created by genetic engineering, and specifically, it refers toa chimeric monoclonal antibody such as a mouse/human chimeric monoclonalantibody, which is characterized as the monoglobulin gene whose variableregion is a mouse immunoglobulin variable region and the constant regionis a human immunoglobulin constant region. The human immunoglobulinconstant region is characterized to have an amino acid sequencedepending on the isotype IgG, IgM, IgA, IgD or IgE, and the constantregion of the recombinant chimeric monoclonal antibody of the inventionmay be the constant region of a human immunoglobulin belonging to any ofthe isotypes. It is preferably the constant region of human IgG. Thechimeric monoclonal antibody of the invention may be produced, forexample, in the following manner. It will be appreciated withoutmention, however, that the production method is not limited to the onedescribed below.

For example, a mouse/human chimeric monoclonal antibody may be preparedwith reference to Jikken Igaku (Experimental Medicine) (special issue)I, Vol. 6, N. 10, 1988 and Japanese Examined Patent Publication HEI No.3-73280. That is, it may be prepared by inserting into one or separateexpression vectors the CH gene (C gene encoding the H chain constantregion), taken from DNA encoding human immunoglobulin, downstream fromthe active VH gene (rearranged VDJ gene encoding the H chain variableregion) taken from DNA encoding the monoclonal antibody isolated fromthe mouse monoclonal antibody-producing hybridoma, and the CL gene (Cgene encoding the L chain constant region) taken from DNA encoding humanimmunoglobulin, downstream from the active VL gene (rearranged VJ geneencoding L chain variable region) taken from DNA encoding the monoclonalantibody isolated from the same hybridoma, with each arranged in anexpressible manner, transforming host cells with the expression vectorand culturing the transformed cells.

Specifically, first a DNA is extracted from the mouse monoclonalantibody-producing hybridoma by a common procedure, and then the DNA isdigested with appropriate restriction endonucleases (for example, EcoRI,HindIII, etc.) and the digested fragments are subjected toelectrophoresis (for example, using a 0.7% agarose gel) for Southernblotting. The electrophoresed gel is stained with ethidium bromide, forexample, and photographed, after the marker positions are attached, andthe gel is washed twice and then immersed for 15 minutes in a 0.15 M HClsolution. It is then immersed for 10 minutes in a 0.4 N NaOH solutionwith gentle shaking. A common method is used for transfer the DNA to afilter, recovery of the filter. After 4 hours, the filter is washedtwice with 2×SSC. After thoroughly drying the filter, it is baked (75°C., 3 hours). Upon completion of baking, the filter is placed in a0.1×SSC/0.1% SDS solution and incubated at 65° C. for 30 minutes. It isthen immersed in a 3×SSC/0.1% SDS solution. The obtained filter isplaced in a plastic bag together with the prehybridization solution, andincubated at 65° C. for 3-4 hours.

Next, 32P-labelled probe DNA and hybridization solution are added forreaction at 65° C. for about 12 hours. After completion ofhybridization, the filter is washed with an appropriate saltconcentration, reaction temperature and time (for example, 2×SSC, 0.1%SDS solution, room temperature, 10 minutes). The filter is placed in aplastic bag, a small amount of 2×SSC is added, the bag is sealed, andautoradiography is performed. This Southern blot method allowsidentification of the rearranged VDJ gene and VJ gene encoding the Hchain and L chain of the mouse monoclonal antibody, respectively. Thezones containing the DNA fragments identified by the method describedabove are fractionated by sucrose density gradient centrifugation andthe isolated DNA is incorporated into a phage vector (for example,charon4A, charon28, λEMBL3, λEMBL4, etc.), and then E. coli (forexample, LE392, NM539, etc.) is transformed with the phage vector, and agenomic library is created. The genomic library is used for plaquehybridization according to the method of Benton and Davis (Science, Vol.196, pp. 180-182 (1977)) using the appropriate probe (H chain J gene, Lchain (κ)J gene, etc.), and the positive clones containing either therearranged VDJ gene or VJ gene are obtained. Restriction enzyme maps ofthe obtained clones are prepared and the nucleotide sequences aredetermined to confirm that the obtained genes contain the targetrearranged VH (VDJ) gene or VL (VJ) gene.

Separately, the human CH gene and human CL gene used for chimerizationare isolated. For example, when creating a chimera with human IgG1, theCγ1 gene as the CH gene and the Cκ gene as the CL gene are isolated. Bytaking advantage of the high homology between the nucleotide sequencesof the mouse immunoglobulin genes and human immunoglobulin genes, thesegenes can be obtained using as probes the mouse Cγ1 gene and mouse Cκgene, which correspond to the human Cγ1 gene and human Cκ gene for theirisolation from a human genomic library.

Specifically, the 3 kb HindIII-BamHI fragment from clone Ig146 (Proc.Natl. Acad. Sci. USA, Vol. 75, pp. 4709-4713 (1978)) and the 6.8 kbEcoRI fragment from clone MEP10 (Proc. Natl. Acad. Sci. USA, Vol. 78,pp. 474-478 (1981)) are used as probes to isolate a DNA fragmentcontaining the human K gene containing the enhancer region, which isderived from a human % Charon4A HaeIII-AluI genomic library (Cell, Vol.15, pp. 1157-1174(1978)). The human Cγ1 gene is isolated, for example,by digesting human embryonic liver cell DNA with HindIII, fractionatingby agarose gel electrophoresis, and inserting the 5.9 kb band in λ788and using the aforementioned probes.

The mouse VH gene and mouse VL gene and the human CH gene and human CLgene obtained in this manner are incorporated into an expression vector,such as pSV2gpt or pSV2neo by a common procedure, using an appropriaterestriction endonuclease and DNA ligase, taking into account thepromoter region and enhancer region, so that the human CH gene is placeddownstream from the mouse VH gene and the human CL gene is placeddownstream from the mouse VL gene. Here, the chimeric genes of the mouseVH gene/human CH gene and mouse VL gene/human CL gene may be arrangedsimultaneously in the same expression vector, or they may be arranged inseparate expression vectors.

The expression vector inserting the chimeric gene constructed in thismanner is then introduced into myeloma cells such as P3X63-Ag8-653 cellsor SP210 cells, which do not of themselves produce antibodies, by theprotoplast fusion method, DEAE-dextrin method, calcium sulfate method,electroporation or the like. The transformed cells are selected out byculturing in medium containing a drug corresponding to the drugresistance gene, which is introduced into the expression vector, and thetarget chimeric monoclonal antibody-producing cells are isolated. Thetarget chimeric monoclonal antibodies are taken from the culturesupernatant of the selected antibody-producing cells.

The “human-type antibodies (CDR-grafted antibodies)” according to theinvention are monoclonal antibodies prepared by genetic engineering, andspecifically, they are human-type monoclonal antibodies characterized inthat all or a portion of the complementarity-determining region of thehypervariable region is the complementarity-determining region of thehypervariable region derived from the mouse monoclonal antibody, theframework region of the variable region is the framework region of thevariable region derived from the human immunoglobulin, and the constantregion is the human immunoglobulin region.

The complementarity-determining regions are the three regions found inthe hypervariable region of the variable region of the antibody, whichare the sites of direct complementary binding to the antigen (CDRs:complementarity-determining regions; CDR1, CDR2, CDR3), and the variableframework regions are the four regions lying before and after the threecomplementarity-determining regions, which are relatively conserved(Framework regions: FR1, FR2, FR3, FR4). Stated differently, this meansa monoclonal antibody wherein all of the regions except for all or aportion of the complementarity-determining region of the hypervariableregion of the mouse monoclonal antibody are repieced by thecorresponding regions of the human immunoglobulin. The constant regionderived from the corresponding region of the human immunoglobulin hasthe amino acid sequence characteristic of each isotype IgG, IgM, IgA,IgD or IgE, and the constant region of the human-type monoclonalantibodies of the invention may be the constant region of humanimmunoglobulin belonging to any isotype, preferably the constant regionof human IgG. There are also no restrictions on the framework regions inthe variable region derived from the human immunoglobulin.

The human-type monoclonal antibodies of the invention may be produced,for example, in the following manner, with understanding that there isno limitation to this production method. For example, the recombinanthuman-type monoclonal antibodies derived from mouse monoclonalantibodies may be prepared by genetic engineering with reference toJapanese Patent Public Inspection HEI No. 4-506458 and JapaneseUnexamined Patent Publication SHO No. 62-296890. That is, at least onemouse H chain CDR gene and at least one mouse L chain CDR gene paring tothe mouse H chain CDR gene are isolated from the mouse monoclonalantibody-producing hybridoma, and a human H chain gene encoding theentire region other than the human H chain CDR corresponding to themouse H chain CDR and the human L chain encoding the entire region otherthan the human L chain CDR corresponding to the mouse L chain CDR areisolated from the human immunoglobulin gene.

The isolated mouse H chain CDR gene and human H chain gene areintroduced in an expressible manner into an appropriate expressionvector, and likewise the mouse L chain CDR gene and the human L chaingene are introduced in an expressible manner into another appropriateexpression vector. Alternatively, the mouse H chain CDR gene/human Hchain gene and the mouse L chain CDR gene/human L chain gene may beintroduced in an expressible manner into the same expression vector. Bytransforming host cells with the expression vector prepared in thismanner, it is possible to obtain human-type monoclonalantibody-producing transformants, and by culturing these transformants,it is possible to obtain the target human-type monoclonal antibodiesfrom the culture supernatant.

A “human antibody” according to the invention is an immunoglobulinwherein all of the regions of the immunoglobulin including the H chainvariable region and H chain constant region and the L chain variableregion and L chain constant region are derived from a gene encoding ahuman immunoglobulin. Human antibodies may be produced by the samemethod used to prepare polyclonal antibodies or monoclonal antibodiesdescribed above. For example, human antibodies are made byimmunosensitization of a transgenic animal, which was created byincorporating at least a human immunoglobulin gene into the gene locusof a non-human mammalian animal, such as a mouse, according to a commonprocedure. For example, a human antibody-producing transgenic mouse canbe created according to the procedure described in Nature Genetics, Vol.7, pp. 13-21, 1994; Japanese Patent Public Inspection HEI No. 4-504365;International Patent Disclosure WO94/25585; Nikkei Science, No. 6, pp.40-50, 1995; Nature, Vol. 368, pp. 856-859, 1994; or Japanese PatentPublic Inspection HEI No. 6-500233.

“Antibody portion” according to the invention means an antibody fragmentcontaining at least one variable region, and refers to a partial regionof an antibody, preferably a monoclonal antibody, according to theinvention mentioned above; specifically, it refers to the Fv, F(ab′)2,Fab′ or Fab fragments. Here, “F(ab′)2” and “Fab′” refer to antibodyfragments produced by treating an immunoglobulin (monoclonal antibody)with a protease such as pepsin or papain, and they are obtained bydigestion before and after the sulfide bonds present between the two Hchains at the hinge region. For example, treatment of IgG with papaincleaves it upstream from the disulfide bonds present between the two Hchains at the hinge region, resulted in producing two homologousantibody fragments, each consisting of an L chain composed of a VL (Lchain variable region) and CL (L chain constant region) and an H chainfragment composed of a VH (H chain variable region) and CHγ1 (γ1 regionof the H chain constant region), which are bonded by sulfide bonds atthe C-terminal region. These two homologous antibody fragments are bothdesignated as Fab′. Treatment of IgG with pepsin cleaves it downstreamfrom the disulfide bonds present between the two H chains at the hingeregion, resulted in producing an antibody fragment which is slightlylarger than the aforementioned two Fab′ fragments connected at the hingeregion. This antibody fragment is designated as F(ab′)2.

(Recombinant Vector and Transformant)

The present invention further provides a recombinant vector containingthe gene or DNA fragment of the invention.

The recombinant vector may be prepared by linking the gene of interestto a recombination vector which is readily available to those skilled inthe art (for example, plasmid DNA or the like) by a common procedure.Examples of vectors to be used include, but are not limited to,pBluescript, pUC18, pUC19 and pBR322, as plasmids derived from E. coli.

An expression vector is particularly useful for the purpose of producinga protein of interest. The type of expression vector is not particularlyrestricted so long as it has the function of expressing the gene ofinterest in host cells, either or both prokaryotic cells and eukaryoticcells, to produce the protein of interest. For example, pQE-30, pQE-60,pMAL-C2, pMAL-p2 and pSE420 are preferred as expression vectors for E.coli, pYES2 (Saccharomyces) and pPIC3.5K, pPIC9K, pAO815 (all of genusPichia) as expression vectors for yeast, and pBacPAK8/9, pBK283, pVL1392and pBlueBac4.5 as expression vectors for insects.

As an example of a method for incorporating a gene fragment of theinvention into a vector such as a plasmid, there may be the proceduredescribed in “Sambrook, J. et al., Molecular Cloning, A LaboratoryManual, (Second edition), Cold Spring Harbor Laboratory, 1. 53 (1989)”.A commercially available ligation kit (for example, by Takara Shuzo) canbe conveniently used. The recombinant vector (recombinant plasmid, forexample) obtained in this manner may be introduced into host cells bythe method described below.

Introduction of a recombinant vector of the invention into host cells(transformation or transfection) can be accomplished by a conventionallyknown protocol, and as examples, there may be the calcium chloridemethod or calcium chloride/rubidium chloride method, electroporation,electroinjection, chemical treatment with PEG or the like, a methodusing a gene gun, etc., as described in “Sambrook, J. et al., MolecularCloning, A Laboratory Manual, (Second edition), Cold Spring HarborLaboratory, 1. 74 (1989)”. Alternatively, the transformation may beaccomplished by the method of Cohen et al. [Proc. Natl. Acad. Sci. USA,69, 2110 (1972)], the protoplast method [Mol. Gen. Genet., 168, 111(1979)] or the competent method [J. Mol. Biol., 56, 209 (1971)], forexample, when the host cells are bacteria (E. coli, Bacillus subtilis,etc.); by the method of Hinnen et al. [Proc. Natl. Acad. Sci. USA, 75,1927 (1978)] or the lithium method [J. Bacteriol., 153, 163 (1983)], forexample, when the host cells are Saccharomyces cerevisiae; by the leafdisk method [Science, 227, 129 (1985)] or the electroporation method[Nature, 319, 791 (1986)], for example, when the host cells are plantcells; by the method of Graham [Virology, 52, 456 (1973)], for example,when the host cells are animal cells; or by the method of Summers et al.[Mol. Cell. Biol., 3, 2156-2165 (1983)], for example, when the hostcells are insect cells.

There are no particular restrictions on the host cells to be used tocreate the transformants so long as they can accommodate and betransformed by the recombinant vector of the invention. Various types ofcells may be used, such as naturally existing cells or artificiallyestablished recombinant cells, as are commonly used in the technicalfield of the invention. For example, there may be mentioned prokaryoticcells such as bacteria (Escherichia, Bacillus) and the like, lowereukaryotic cells including monocellular hosts such as yeast(Saccharomyces, Pichia) and the like, and higher eukaryotic cells suchas silkworm cells and the like. The host cells are preferably E. coli,yeast or insect cells, with specific examples including E. coli (M15,JM109, BL21, etc.), yeast (INVSc1 (Saccharomyces), GS115, KM71 (both ofPichia), etc.), and insect cells (BmN4, silkworm larva, etc.). Examplesof animal cells include mouse-derived, Xenopus laevis-derived,rat-derived, hamster-derived, monkey-derived and human-derived cells, orcultured cell lines established from these cells.

When the host cells are bacteria, particularly E. coli, they willusually include at least the expression vector, which has thepromoter/operator region, an initiation codon, the gene encoding theprotein of interest, a termination codon, a terminator and a replicableunit. When the host cells are yeast, plant cells, animal cells or insectcells, they will usually include at least the expression vector andpreferably a promoter, initiation codon, the gene encoding the proteinof interest, a termination codon and a terminator. As appropriate, theymay also contain, DNA encoding the signal peptide, an enhancer sequence,the non-translated regions at the 5′ and 3′ ends of the gene ofinterest, a selection marker region or a replicable unit.

The preferred initiation codon for the vector of the invention is themethionine codon (ATG). Examples of termination codons are the ordinarytermination codons (for example, TAG, TGA and TAA).

A replicable unit means DNA with the ability to replicate its entire DNAsequence in the host cells, and this includes natural plasmids,artificially modified plasmids (plasmids prepared from natural plasmids)and synthetic plasmids. As preferred plasmids, there may be plasmidspQE30, pET and pCAL or their artificially modified forms (DNA fragmentsobtained by treating pQE30, pET or pCAL with an appropriate restrictionendonuclease), for E. coli, plasmids pYES2 and pPIC9K for yeast orplasmid pBacPAK8/9 for insect cells.

The enhancer sequence and terminator sequence used may be ones commonlyused by those skilled in the art, such as the ones derived from SV40.The selection marker may be a common one used following an ordinarymethod. Examples thereof include resistance genes against antibiotics,such as tetracycline, ampicillin, kanamycin, neomycin, hygromycin,spectinomycin or chloramphenicol.

The expression vector may be prepared by linking the aforementionedpromoter, initiation codon, gene encoding the protein of interest,termination codon and terminator region in a continuous and cyclicmanner in the appropriate replicable unit. Here the appropriate DNAfragments (for example, linkers, other restriction enzyme sites, etc.)may be used following a common procedure, such as digestion with arestriction enzyme and ligation using T4DNA ligase, as desired.

(Receptor, Screening Method, Novel Substance)

The protein encoded by the gene of the invention may possibly act at theentry point of induction and stimulation of G-CSF. (That is, while thepresent invention is not restricted in any way by the following theory,one possible explanatory model is that binding of an external ligand tothe protein of the invention residing on the surface layer of macrophagecells, transmitting of the resulting signal into the cell, and leadingto release of G-CSF by the macrophage.) Consequently, the protein of theinvention could be the receptor working as a granulocytecolony-stimulating factor-inducer or a portion thereof. A “portion of areceptor” would include a subunit of the receptor, possibly modifiedwith a sugar chain or the like. The receptor has the ability to bind(also known as “affinity”) to substances that can allow to induceproduction of granulocyte colony-stimulating factor, such as monoclonalantibodies produced by the hybridoma deposited as FERM BP-6103 or theirfragments, and it may possibly reside in the cell membrane of cellscapable of producing granulocyte colony-stimulating factor, includingmacrophages. The present invention provides such a receptor.

The invention further provides a useful substance screening method byusing the protein of the invention. Such a screening method includesmeasurement of binding between the substance of interest and the proteinof the invention, or the receptor, measurement of the effect of thesubstance of interest via the receptor (for example, production of G-CSFby the macrophages or production of a marker substance fromappropriately transformed cells), or comparison between the structure ofthe substance of interest (for example, its amino acid sequence when thesubstance of interest is a protein) and the structure of the protein ofthe invention (for example, its amino acid sequence).

The protein of the invention used for screening is preferably (a) aprotein having the amino acid sequence listed as SEQ ID NO:4 of theSequence Listing; (b) a protein having the amino acid sequence listed asSEQ ID NO:4 of the Sequence Listing with one or more amino aciddeletions, substitutions, additions or insertions and also binding to anantibody or its fragment that is active to induce granulocytecolony-stimulating factor; (c) a protein having at least 50% (preferablyat least 60%, more preferably at least 70%, even more preferably atleast 80%, even more preferably at least 90%, especially preferably atleast 94% and most preferably at least 98%) homology with the amino acidsequence listed as SEQ ID NO:4 and also binding to an antibody or itsfragment that is active to induce granulocyte colony-stimulating factor;or (d) a protein that is encoded by DNA which hybridizes with DNA havingthe nucleotide sequence listed as SEQ ID NO:3 of the Sequence Listingunder stringent conditions and that binds to an antibody or its fragmentthat is active to induce granulocyte colony-stimulating factor.

The following is a more specific example of the screening method: Avector is constructed by inserting the G-CSF promoter gene and a geneencoding a marker protein, such as luciferase, β-galactosidase, GreenFluorescent Protein (GFP), β-lactamase or chloramphenicolacetyltransferase (CAT), at downstream thereof, and a drug resistancegene against a drug, such as tetracycline, ampicillin, kanamycin,neomycin, hygromycin or spectinomycin, at further downstream thereof.The vector is introduced into cells (for example, a macrophage cellline, and preferably a human-derived macrophage cell line) bearingreceptors, which contains the protein of the invention. The obtainedcells are treated with a drug-containing medium, and colony-formingcells are selected. Clones expressing the marker protein upon inductionare then selected. It is then confirmed that expression of the markerprotein reflects actual expression of G-CSF mRNA. The transformed cellline obtained in this manner is treated with various substances,followed by screening for substances that have induced expression of themarker protein.

A useful substance obtained by screening is (a) a substance which canbind to the receptor, and as a result of its binding to the receptor, itcan elicit a structural change in the receptor, transmit signals intothe cell via the receptor, and induce production of granulocytecolony-stimulating factor (also known as an “agonist” or “agent”); (b) asubstance which can bind to the receptor, and as a result of its bindingto the receptor, it can inhibit the binding of the receptor to thesubstances that can induce production of granulocyte colony-stimulatingfactor, but it in itself does not induce production of granulocytecolony-stimulating factor (also known as an “antagonist” or “blocker”);or (c) a substance which can bind to the receptor, and as a result ofits binding to the receptor, it can inhibit the binding of the receptorto the substances that can induce production of granulocytecolony-stimulating factor, but it in itself blocks production ofgranulocyte colony-stimulating factor (also known as an “inverseagonist” or “reagent”).

Such substances are novel. Thus, the present invention also provides (a)a substance which can bind to the receptor, and as a result of itsbinding to the receptor, it can induce a change in the receptor,transmit signals into the cell via the receptor, and induce productionof granulocyte colony-stimulating factor; (b) a substance which can bindto the receptor, and as a result of its binding to the receptor, it caninhibit the binding of the receptor to the substances that can induceproduction of granulocyte colony-stimulating factor, but it in itselfdoes not induce production of granulocyte colony-stimulating factor; and(c) a substance which can bind to the receptor, and as a result of itsbinding to the receptor, it can inhibit the binding of the receptor tothe substances that can induce production of granulocytecolony-stimulating factor, but it in itself blocks production ofgranulocyte colony-stimulating factor, which are obtained by the methoddescribed above. The invention still further provides a substance whichcan bind to the receptor, and (a) as a result of its binding to thereceptor, it can induce a change in the receptor, transmit signals intothe cell via the receptor, and induce production of granulocytecolony-stimulating factor; (b) as a result of its binding to thereceptor, it can inhibit the binding of the receptor to the substancesthat can induce production of granulocyte colony-stimulating factor, butit in itself does not induce production of granulocytecolony-stimulating factor; or (c) as a result of its binding to thereceptor, it can inhibit the binding of the receptor to the substancesthat can induce production of granulocyte colony-stimulating factor, butit in itself inhibits production of granulocyte colony-stimulatingfactor. Such substances will hereinafter be referred to as “substancesof the invention”.

Examples of substances of the invention include the antibody of theinvention, its fragments and other low molecular compounds, among whichthere are those with the effect of inducing production of granulocytecolony-stimulating factor, those with the effect of inhibition ofreceptor binding to the substances that can induce production ofgranulocyte colony-stimulating factor, and those with the effect ofinhibition of receptor binding to the substances that can induceproduction of granulocyte colony-stimulating factor, while those alsoinhibit production of granulocyte colony-stimulating factor.

When the substance of interest is an antibody, the binding to thereceptor (or binding inhibition) can be measured by a method, such as,for example, analysis of the antibody-bound macrophage cells using flowcytometry or ELISA.

Inducing effect (or inhibiting effect) of the production of granulocytecolony-stimulating factor can be determined by the method described inJapanese Unexamined Patent Publication HEI No. 11-106400. The outline ofthe process is given below.

The G-CSF promoter gene is inserted between the XhoI and the NcoI siteof PicaGene Enhancer Vector 2 (product of Wako Junyaku Kogyo Co., Ltd.),in order to construct vector Pica G-CSF neo, the luciferase gene islinked downstream therefrom in place of the G-CSF gene itself, and thena neomycin resistance gene cut out from pMC1Neo PolyA at the SalI sitedownstream from SV40 also. This vector is introduced into the RAW264.7cells by electroporation. The obtained cells are treated with mediumcontaining geneticin, and the colony-forming cells are selected. Amongthe geneticin-resistant clones, clones exhibiting luciferase activityupon induction are further selected. Northern blot analysis using32P-labeled mouse G-CSF cDNA as the probe is made to confirm that theluciferase activity reflects actual expression of the G-CSF mRNA. Thetransformed macrophage cells obtained in this manner are plated in a96-well microtiter plate at 5×104 cells per well and cultured at 37° C.for 24 hours, and then after treating them with a prepared agonist orantagonist, as need, or the substance of interest is added atconcentrations of about 0, 3.75, 7.5, 15, 30 and/or 60 μg/ml. Afterfurther culturing at 37° C. for 18 hours, the luciferase activity ismeasured.

(Use of Gene of the Invention as Drug Agent)

The gene of the invention may be utilized, for example, for diagnosis,prevention and therapy (gene therapy, etc.) of diseases in whichneutrophils, a type of blood leukocyte, are involved (such asneutropenia). The protein, a part of the protein or the peptidesthereof, antibody or its fragment, receptor, or the substance of theinvention (hereinafter these will sometimes be referred to collectivelyas “protein, etc. of the invention”) can serve as a drug to regulate thenumber of neutrophils in the blood or bone marrow. That is, the gene andprotein, etc. of the invention can be used for treatment of neutropeniaas a side effect of anticancer agents or neutropenia directly followingbone marrow transplantation, and for diagnosis, prevention and treatmentof anaplastic anemia.

The protein, etc. of the invention may generally be administeredsystemically or locally, usually in a parenteral form. Intravenousadministration is particularly preferred for the parenteral forms.

The gene of the invention may be administrated systemically or locallyin the form of “gene therapy”, wherein the gene is introduced into cellseither in vivo or ex vivo. Introduction of the gene can be accomplished,for example, by the method described in Biomanual UP Series, IdenshiChiryo no Kiso Gijutsu [Fundamental Techniques for Gene Therapy],Shimada, T., Saito, I., Ozawa, T., ed.: Yodosha Publishing, 1996. Forintroduction into cells ex vivo, there may be methods employing aretrovirus vector, adenovirus vector, adeno-associated virus (AAV)vector, cationic liposomes, HVJ-liposomes, or the calcium phosphatemethod, DEAE dextran method, etc. For introduction into cells in vivo,there may be methods employing a retrovirus vector, adenovirus vector,adeno-associated virus (AAV) vector, cationic liposomes orHVJ-liposomes.

The administration dosage will differ depending on age, gender, bodyweight, symptoms, treatment effect, administration route, treatment timeand substance administrated (types of the protein or gene). Butparenteral administration one to several times per day may be given at adosage in the range of 1 μg to 100 g and preferably in the range of 10μg to 1000 mg for each time for adults. Since the administration dosagewill vary depending on the conditions, a dosage below this range willoften be sufficient, or a dosage exceeding this range may be necessary.Injections for parenteral administration according to the inventioninclude sterile aqueous or nonaqueous solutions, suspensions andemulsions. For aqueous and nonaqueous solutions and suspensions, one ormore active substances are mixed with at least one inactive dilutingagent. As examples of aqueous diluting agents, there may be distilledwater for injection and physiological saline. As examples of nonaqueousdiluting agents, there may be propylene glycol, polyethylene glycol,vegetable oils, such as olive oil, and alcohols, such as ethanol.

Such a composition may also include adjuvants, such as preservatives,humectants, emulsifiers, dispersers and stabilizers (for example,arginine and aspartic acid).

These can be sterilized by passage through a bacteria capturing filter,mixture with a sterilizer or irradiation. They may also be prepared assterile solid compositions by lyophilization, for example, and thendissolved in sterile distilled water for injection or another solventprior to use.

Other compositions for parenteral administration include externalapplications or suppositories and pessaries for enteric administration,which are formulated according to ordinary methods and contain one ormore active substances.

The invention will now be explained in greater detail by way of thefollowing examples, with understanding that the invention is in no wayrestricted by these examples.

EXAMPLES Example 1 Cloning of Antigen Gene Recognized by the MonoclonalAntibody from Macrophage Cell Line

(1) Preparation of poly(A)⁺ RNA from Macrophage Cells (RAW264.7)

Guanidium thiocyanate-phenol-chloroform single-step extraction(Laboratory Manuals of Genetic Engineering, 3rd Edition, pp. 83-84,1996) was used to prepare approximately 0.3 mg of total mRNA from 2×10⁸mouse macrophage cells (RAW264.7). This was purified using an oligo(dT)cellulose column to obtain 5 μg of poly(A)⁺ RNA.

(2) Synthesis of Double-Stranded cDNA from poly(A)⁺ RNA

A reaction solution (50 μl) containing the poly(A)⁺ RNA as obtainedabove (1) (5 μg), a reverse transcriptase (MMLV-RTase; product ofSTRATAGENE Corp.; 70 units) and dNTPs (0.6 mM) was incubated at 37° C.for 60 minutes to synthesize of a first strand cDNA. Next, a reactionsolution containing the aforementioned reaction solution (45 μl), DNApolymerase (product of STRATAGENE Corp.; 100 units) and dNTPs (0.3 mM)was incubated at 16° C. for 150 minutes to synthesize a second strandcDNA, to obtain double-stranded cDNA (8 μg).

(3) Construction of cDNA Library

The method of Gubler-Hoffmann (Gubler, U. and Hoffmann, B. J.: Gene,25:263-269, 1983) was used PfuDNA polymerase was used to blunt the endsof the double-stranded cDNA as synthesized above (2), and an adapter waslinked thereto, by ligation with T4DNA ligase. Specifically, a reactionsolution (total: 225 μl) containing the double-stranded cDNA as obtainedabove (2) (8 μg DNA; 200 μl) and PfuDNA polymerase (5 units) wasincubated at 72° C. for 30 minutes.

The adapter-linked DNA was cleaved at the ends with restriction enzymeXhoI, and the cDNA longer than 0.5 kbp was fractionated with a gelcolumn. This cDNA was incorporated into XZAPII phage vector (STRATAGENECorp.) with T4DNA ligase following common protocol, and then by ligationinto phage particles. Measurement of the phage titer showed that thecDNA library contained 2×10⁶ independent clones. The obtained phagelibrary was used to infect E. Coli (XL1-Blue MRF′) and was allowed toproliferate to 3.4×10⁹ pfu/ml.

(4) Screening of Genes Encoding Protein that Bind to Antibodies thatShow the Ability to Induce Granulocyte Colony-Stimulating Factor

The cDNA library as constructed above (3) was subjected toimmunoscreening using monoclonal antibodies (produced by the hybridomadeposited as FERM BP-6103; described in Japanese Patent Application HEINo. 9-266591), which shows the ability to induce granulocytecolony-stimulating factor (G-CSF) as the prove. The specific procedurewas as follows.

E. coli (XL1-BlueMRF′) infected with the phage cDNA library was seededonto a 150 mm diameter plate. The plate was incubated at 42° C. for 4hours that allowed the formation of the plaques of approximately 0.5 mmdiameter. Then, microcellulose membranes, which were immersed in 10 mMof IPTG (isopropylthio-β-galactoside) and air-dried, were placed onthose plates and incubated for 3 hours at 37° C. The nitrocellulosemembrane was peeled off, and incubated in TBS-T (20 mM Tris-HCl, pH 7.6,0.1% Tween20) containing 5% skim milk for one hour for blocking themembranes, while agitating at room temperature. This was followed byrinsing of the membrane gently for 2 minutes with TBS-T (repeatedtwice), immersing in a buffer at room temperature for 15 minutes (once)and washing for 5 minutes (twice). The nitrocellulose membrane wasincubated for one hour at room temperature for reaction with theantibodies while agitating in the diluted primary antibody solution(produced by the hybridoma deposited as FERM BP-6103; 1.6 μg/ml). Thenitrocellulose membrane was then washed in the same manner as theprevious washing. Alkaline phosphatase-labeled secondary antibodies(ZYMED) were diluted to 0.6 μg/ml with TBS containing 1% BSA, and thenitrocellulose membrane was incubated for one hour at room temperaturefor reaction with the antibodies, while agitating in the dilutedsecondary antibody solution. The nitrocellulose membrane was againthoroughly washed in the same manner as described above, and finallywashed with TBS for 5 minutes. After adding 1 ml of each NBT solution(50 mg/ml of NitroBlue Tetrazolium in 70% dimethylformamide) and BCIPsolution (50 mg/ml of 5-bromo-4-chloro-3-indolyl phosphate indimethylformamide) to a buffer solution containing 100 mM of Tris-HCl(pH 9.5), 100 mM of NaCl and 5 mM of MgCl₂, the nitrocellulose membranewas immersed therein. Reaction was carried out for 30 minutes in a darkroom, and the membrane washed with water and dried. After drying, theplaques exhibiting positive reaction on the nitrocellulose membrane werecollected from the original plate.

Finally, 22 antibody-binding positive clones were obtained from 7×10⁵phage in the first screening. The top agarose containing the positiveplaques were collected, and amplified. The second, third and fourthscreenings were performed from approximately 1000 phages following thesame procedure described above, resulting in isolation of 3 positiveclones (MMR10, MMR17 and MMR19).

(5) Determination of the Nucleotide Sequence of the Gene

The inserts of the 3 positive clones (MMR10, MMR17 and MMR19), asobtained above (4) were cut out from the λZAPII phage vectors by in vivoexcision following a common procedure, and subcloned by converting thevector to pBluescript SK(−) Phagemid. The subcloned plasmids were madelarge quantity in E. coli (SOLR), and approximately 20 μg of the plasmidDNA was obtained. The Primer Walking method was used to analyze thenucleotide sequences of these DNA.

Based on the results of the nucleotide sequence analyses, a clone MMR19was found to have the 840 bp nucleotide sequence of the full length cDNAwhich included the open reading frame of the protein. The nucleotidesequence of the clone MMR19 is listed as SEQ ID NO:1 of the SequenceListing.

(6) Primary Structure of the Protein Deduced from the NucleotideSequence of cDNA Clone

The primary structure of the protein (MMR-CAM) (listed as SEQ ID NOS:1 &2 in the Sequence Listing) deduced from the nucleotide sequence of thegene analyzed (5) consists of 241 amino acid residues, and the molecularweight as estimated from the amino acid sequence was approximately 27kDa. MMR-CAM is thought to be a type I membrane glycoprotein with onemembrane-spanning domain, which comprises an extracellular portion of107 amino acids, a membrane-spanning portion of 23 amino acids and anintracellular portion of 111 amino acids. Homology search showed that nomolecules were found similar to the protein of the invention in terms ofthe structure, suggesting that the protein of the invention does notbelong to the existing family. Also, there is a portion with theextensive modifications by the type O sugar chains was present in theextracellular domain. Phosphorylation sites for protein kinase C,tyrosine kinase, etc. are present in the intracellular domain. Thesesugar chain binding sites and phosphorylation sites are believed to playon very important role in signal transduction.

Example 2 Expression of the Protein (MMR-CAM) of the Invention

The clone (MMR19) obtained in Example 1(4) was inserted into anexpression vector (λZAPII) following a common procedure and transformedE. coli (XL1-Blue), then a transformant cell line was constructed. Thetransformed E. coli cells were cultured, and the culture supernatant wasdot blotted and allowed to with the same monoclonal antibodies used asshown in (3), which was produced by hybridoma deposited as FERM BP-6103as a probe. Following this process, it was confirmed that the culturesupernatant contained the protein that bound to the monoclonalantibodies.

Example 3 Comparison of the Mouse-Derived Protein with Other HomologousProteins Using Database Search

A data-search was conducted for human genes homologous to the nucleotidesequence and amino acid sequence listed as SEQ ID NO:1 and determined inExample 1 on both the amino acid level and the DNA level databases using(DNA DATA BANK of JAPAN (DDBJ): Dept. of Education, National Instituteof Genetics, Center for Information Biology). The results are shown inTables 1 and 2. These results suggest that the gene of the invention isalso conserved in humans with high homology. TABLE 1 Homology on aminoacid level Position within amino acid Matching in sequence of SEQ ID NO:1 human homologue  1 to 91 83/91 (91%)  50 to 146 83/97 (85%)  1 to 7870/78 (89%) 200 to 241 40/42 (95%) 172 to 241 67/70 (95%) 103 to 15046/48 (95%) 169 to 241 58/73 (79%)

TABLE 2 Homology on DNA level Position within nucleotide Matching insequence of SEQ ID NO: 1 human homologue 519 to 736 189/218 (86%) 666 to689  23/24 (95%) 381 to 403  22/23 (95%) 709 to 727  19/19 (100%)

Example 4 Cloning of the Human Homologue of the Antigen Gene

Guanidium thiocyanate-phenol-chloroform extraction was used to extracttotal RNA from human normal brain tissue, and the poly(A)+ RNA waspurified using oligo(dT) cellulose. cDNA was synthesized from thePoly(A)+ RNA using reverse transcriptase (MMLV-RTase) and DNApolymerase. A sense primer of position 4 to 22 (CCATGTCTGGCTGTCAAGC (SEQID NO:5)) and an antisense primer of position 714 to 724(CCATTTTCTCCAACTGGGAGC (SEQ ID NO:6)) of the mouse antigen gene (MMR19)sequence were prepared, and these primers and the human normal braintissue cDNA as the template were used for PCR reaction. As a result, apartial cDNA of the human homologue of the mouse antigen gene (MMR19)was obtained. Next, the 3′RACE method and 5′RACE method were carried outusing a specific primer (GSP) for the human homologue partial cDNA andan adapter primer. An antisense primer (GTCAGAAGAGATTCAGGGTGACC (SEQ IDNO:7)) was prepared from the 3′ RACE fragment and a sense primer(AAGCCGTGCGGAGATTGGAGG (SEQ ID NO:8)) from the 5′ RACE fragment. As aresult of LD-PCR, the full length cDNA of the human homologue includingthe open reading frame was obtained. The Primer Walking method was usedto elucidate the 924 bp nucleotide sequence of the cDNA. The obtainednucleotide sequence is listed as SEQ ID NO:3 of the Sequence Listing.The nucleotide sequence of the human homologue cDNA (924 bp) showed84.8% homology (with 712 matching nucleotides out of 924) with thenucleotide sequence of the mouse antigen gene cDNA (840 bp).

The primary structure of the protein deduced from the nucleotidesequence of the obtained gene is listed as SEQ ID NOS:3 & 4, consistingof 242 amino acids. The deduced amino acid sequence showed 93.8%homology with the mouse form (with 226 matching residues out of 242).This protein is also thought to be a type I membrane glycoprotein withone membrane-spanning domain.

EFFECT OF THE INVENTION

The gene and the protein encoding by the gene (including fragments ofthe gene and fragments of the protein), antibody (including fragmentsthereof), receptor and substance of the invention are novel, and areuseful for pharmaceutical purposes.

The gene and the protein encoding by the gene (including fragments ofthe gene and fragments of the protein), antibody (including fragmentsthereof) and receptor are also useful as analytical reagents forscreening of substances (for example, monoclonal antibodies, proteinsand other low molecular substances) that have the ability to inducegranulocyte colony-stimulating factor.

Fragments of the gene of the invention are also useful as probes forscreening of homologous genes derived from other organisms.

1. A gene encoding: (a) a protein having the amino acid sequence listed as SEQ ID NO:4 of the Sequence Listing; (b) a protein having the amino acid sequence listed as SEQ ID NO:4 of the Sequence Listing with one or more amino acid deletions, substitutions, additions or insertions and also binding to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor; or (c) a protein having at least 50% homology with the amino acid sequence listed as SEQ ID NO:4 and also binding to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor.
 2. A gene having: (a) the nucleotide sequence listed as SEQ ID NO:3 of the Sequence Listing; (b) a nucleotide sequence which is the nucleotide sequence listed as SEQ ID NO:3 of the Sequence Listing with one or more nucleotide deletions, substitutions, additions or insertions and which encodes a protein that can bind to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor; or (c) a nucleotide sequence which hybridizes with DNA having the nucleotide sequence listed as SEQ ID NO:3 of the Sequence Listing under stringent conditions and encodes a protein that can bind to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor.
 3. A DNA fragment containing: (1) the nucleotide sequence from position 519 to position 736, the nucleotide sequence from position 666 to position 689, the nucleotide sequence from position 381 to position 403 or the nucleotide sequence from position 709 to position 727 of the nucleotide sequence listed as SEQ ID NO:1 of the Sequence Listing; (2) a nucleotide sequence which is any of the nucleotide sequences of (1) with one or more nucleotide deletions, substitutions, additions or insertions; or (3) a nucleotide sequence which has at least 80% homology with any of the nucleotide sequences of (1).
 4. A gene containing: (1) the nucleotide sequence from position 519 to position 736, the nucleotide sequence from position 666 to position 689, the nucleotide sequence from position 381 to position 403 or the nucleotide sequence from position 709 to position 727 of the nucleotide sequence listed as SEQ ID NO:1 of the Sequence Listing; (2) a nucleotide sequence which is any of the nucleotide sequences of (1) with one or more nucleotide deletions, substitutions, additions or insertions; or (3) a nucleotide sequence which has at least 80% homology with any of the nucleotide sequences of (1), and encoding a protein that can bind to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor.
 5. Any of the following proteins: (a) a protein having the amino acid sequence listed as SEQ ID NO:4 of the Sequence Listing; (b) a protein having the amino acid sequence listed as SEQ ID NO:4 of the Sequence Listing with one or more amino acid deletions, substitutions, additions or insertions and also binding to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor; (c) a protein having at least 50% homology with the amino acid sequence listed as SEQ ID NO:4 and also binding to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor; or (d) a protein that is encoded by the DNA which hybridizes with DNA having the nucleotide sequence listed as SEQ ID NO:3 of the Sequence Listing under stringent conditions and that binds to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor.
 6. A protein comprising any of the followings: (1) the amino acid sequence from residues 1 to 91, the amino acid sequence from residues 50 to 146, the amino acid sequence from residues 1 to 78, the amino acid sequence from residues 200 to 241, the amino acid sequence from residues 172 to 241, the amino acid sequence from residues 103 to 150 or the amino acid sequence from residues 169 to 241 of the amino acid sequence listed as SEQ ID NO:2 of the Sequence Listing; (2) an amino acid sequence which is any of the amino acid sequences of (1) with one or more amino acid deletions, substitutions, additions or insertions; or (3) an amino acid sequence having at least 70% homology with any of the amino acid sequences of (1).
 7. A protein comprising any of the followings: (1) the amino acid sequence from residues 1 to 91, the amino acid sequence from residues 50 to 146, the amino acid sequence from residues 1 to 78, the amino acid sequence from residues 200 to 241, the amino acid sequence from residues 172 to 241, the amino acid sequence from residues 103 to 150 or the amino acid sequence from residues 169 to 241 of the amino acid sequence listed as SEQ ID NO:2 of the Sequence Listing; (2) an amino acid sequence which is any of the amino acid sequences of (1) with one or more amino acid deletions, substitutions, additions or insertions; or (3) an amino acid sequence having at least 70% homology with any of the amino acid sequences of (1), and also binding to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor.
 8. An antibody against a protein according to claim 5, or a fragment thereof.
 9. An antibody or fragment thereof according to claim 8, which is a monoclonal antibody.
 10. An antibody or fragment thereof according to claim 9, which is a human-type monoclonal antibody or human monoclonal antibody.
 11. Any of the following substances obtained by a screening method, which is characterized by measurement of binding between a substance and a protein according to claim 5: (a) a substance which can bind to a protein according to claim 5, and as a result of its binding to the protein, it can cause a change in the protein structure, transmit signals into the cell via the protein, and induce production of granulocyte colony-stimulating factor; (b) a substance which can bind to a protein according to claim 5, and as a result of its binding to the protein, it can inhibit the binding of the protein to the substances that can induce production of granulocyte colony-stimulating factor, but it in itself does not induce production of granulocyte colony-stimulating factor; or (c) a substance which can bind to a protein according to claim 5, and as a result of its binding to the protein, it can inhibit the binding of the protein to the substances that can induce production of granulocyte colony-stimulating factor, but it in itself blocks production of granulocyte colony-stimulating factor.
 12. Any of the following substances that can bind to a protein according to claim 5: (a) a substance which, as a result of its binding to the protein, it can cause a change in the protein structure, transmit signals into the cell via the protein, and induce production of granulocyte colony-stimulating factor; (b) a substance which, as a result of its binding to the protein, it can inhibit the binding of the protein to the substances that can induce production of granulocyte colony-stimulating factor, but it in itself does not induce production of granulocyte colony-stimulating factor; or (c) a substance which, as a result of its binding to the protein, it can inhibit the binding of the protein to the substances that can induce production of granulocyte colony-stimulating factor, but it in itself blocks production of granulocyte colony-stimulating factor.
 13. A pharmaceutical composition comprising a gene of claim 1 or 2, a DNA fragment of claim 3, or a protein of claim 5, which is used for diagnosis, prevention or treatment of a disease or condition related to G-CSF, such as infection or neutropenia.
 14. A method of diagnosis, prevention or treatment of a disease or a condition related to G-CSF, such as infection or neutropenia, which employs a gene or DNA fragment according to any one of claims 1 to 4, a protein according to any one of claims 5 to 8, or an antibody or its fragment according to any one of claims 9 to
 10. 